Hydrogel from bovine decellularized ovarian extracellular matrix supports mouse follicle survival in vitro

摘要

Introduction: In case of certain types of cancer, such as leukemia and ovarian cancer, grafting of frozen/thawed ovarian tissue cannot be performed due to the risk or reintroduction of malignant cells. An alternative to restore fertility in these patients is the development of an artificial ovary. To provide an artificial matrix as close as possible to follicle natural environment, we produced and characterized a hydrogel derived from bovine decellularized ovarian extracellular matrix (boECM). Materials and Methods: Bovine ovarian tissue was collected at the local slaughterhouse and was reduced to small pieces. It was then decellularized as described previously. The resulting ECM was lyophilized and then digested with pepsin (1 mg/ml) for 72 hours. A boECM hydrogel was obtained following pH neutralization of the digested solution and incubation at 37°C. boECM hydrogel gelation kinetics and modulus were analyzed by turbidimetry and theology, respectively. The total protein content was measured by BCA and collagen content by a Sircol assay. Gel network morphology was studied by SEM. Finally, the impact of follicle incorporation in the boECM hydrogel was studied in vitro. Three SCID mice were ovarectomized and their ovaries were mechanically disrupted in order to isolate primordial/primary (PP) and secondary (S) follicles. In total, 233 follicles were encapsulated (PP=187; S=46) into boECM (about 10 follicles/bead). Follicle morphology (hematoxilin eosin staining), viability (Live/dead kit assay) and follicle diameter were evaluated at day 0 and day 7. Results and Discussion: Rheological analysis showed that a boECM with an elastic modulus of 60Pa could be obtained after 5 minutes; complete gelation was obtained after 20 minutes. The total protein content was 4mg/ml, whereas the collagen content was 0,6mg/ml. SEM analysis showed moderately organized collagen fibrils with large pores. Concerning follicle in vitro culture, 78% and 57% of PP and S follicles respectively were found viable at day 0. The follicle diameter (mean ± SD) was 36μm ± 10 and 81μm ± 24 for PP and S follicles respectively. At day 7, the survival rate was 54% (101/187) and 63% (29/43) for PP and S follicles respectively. Sixty-eight percent and 76% of follicles were viable in the PP and S group after one week of IVC. The follicle diameter (mean ± SD) was 36μm ± 12 and 73μm + 25 for PP and S follicles respectively. Despite the high survival rate of mouse preantral follicles, no significant follicle growth was observed in any of the follicular categories (primordial/primary and secondary). Conclusion: For the first time, thermosensitive hydrogel derived from decellularized bovine ovarian tissue could be obtained. This boECM hydrogel was completely gellfied after 20 minutes, due to the crosslinking of collagen fibers. It seems to be a promising bio-scaffold to support the survival of isolated murine preantral follicles. However, longer periods of in vitro culture are necessary to assess the effect of the hydrogel on follicle growth.